In the course of development, many genes that are repressed by Polycomb mechanisms in the early embryo or in stem cells, become active to mediate pathways of differentiation. Trithorax and ASH1 proteins are necessary to switch genes to an epigenetic active state and to antagonize Polycomb silencing. While Trithorax is also required for transcription elongation, little is known about how TRX and ASH1 are recruited to their antirepressive function. The mammalian Trithorax homologue, MLL is known to control Hox gene expression and cyclin genes and is a target of chromosomal rearrangements that produce fusions with other proteins, resulting in mixed lineage leukemias. New microarray data in Drosophila reveal that a new kind of TRX/ASH1 complex is implicated in this role and forms an extended domain over Polycomb target genes to maintain their derepressed state. The exceptional extended distribution of these complexes is striking and suggests a functional link between Polycomb Response Elements and transcriptional activity. To understand the nature of these complexes and their function in resetting the epigenetic state of the target genes, TRX andASH1 complexes will be studied in Drosophila, where the relationship between Polycomb Response Elements and their associated genes is better understood. The dynamics of TRX and ASH1 complexes will be studied by chromatin immunoprecipitation and genomic tiling microarrays, using cultured cell lines representing different epigenetic states, as well as Drosophila embryos and larvae. Associated chromatin modifications will be studied together with the effect of loss of function in the trx and ash1 genes. The functional relationship between Polycomb Response Elements and the formation of TRX/ASH1 domains will be studied using reporter transgene constructs. These will permit the dissection of the effect of topology, timing and epigenetic history on the dynamics of TRX/ASH1 domain formation. The nature and composition of the complexes will be determined by purification using biochemical and affinity methods and their histone modification activities will be determined both in vivo and in vitro. The proteolytic processing of the Trithorax protein is necessary for function and is due to a specific protease, Taspase1, conserved in flies and mammals. Only the N-terminal fragment of Trithorax is co-localized with ASH1, giving a new significance to the Taspase function. The Taspase function will be characterized by genetic studies, its chromatin distribution and its association with other components will be determined. Together, these experiments will elucidate the way epigenetic states are switched when cells embark in a program of differentiation and will help to understand the way TRX/MLL fusions can lead to leukemias. PUBLIC HEALTH RELEVANCE: Trithorax complexes are important to support the active state of genes regulated by Polycomb repressive mechanisms in development and differentiation. Inappropriate targeting of human Trithorax produces mixed lineage leukemias. This project will study how TRX and ASH1 complexes are targeted to maintain the active state of their target genes.